Category Archives: General

Wednesday Links

Wednesday Links

hysteria
GMOs

The environmental benefits of genetically modified crops is explored in Conservation Tillage, Herbicide Use, and Genetically Engineered Crops in the United States: The Case of Soybeans

A piece on the claim that GMOs are under-studied, With 2000+ global studies affirming safety, GM foods among most analyzed subjects in science pretty much demonstrates that no, they are not.

Neonicotinoid pesticides are sprayed on crops, and they are bad for good insects. But they’re good for selling plants. Engineered pest resistance doesn’t get sprayed and affects only pests that attack the specific crops. Just sayin’.

Organic foods may have been sprayed with pesticides, too – and isn’t necessarily any better for you. Being free of GMOs makes no difference.

Vaccines

A friend and I were blocked from commenting on an online discussion on the terrible, horrible things that are vaccines. This is a typical technique of anti-vaxxers. A detailed description of what it means to be anti-vaccine is on Science-Based Medicine It’s from 2010, but classics never get old.

Because of a new study analyzing the actual risks of vaccination (hint – nearly none, even less compared with disease outcomes) the pro-vaccine message is finally getting the press it deserves. USA Today, The Daily Beast, Think Progress (I know, not a big anti-vaxxer magnet) The New York Times and Time. Even The Economist reminds us that we should take our medical advice from science, not celebrities.

Medicine

Viruses may be responsible for several cancers. The Big Idea That Might Beat Cancer and Cut Health-Care Costs by 80 Percent explores a virus that may trigger certain kinds. Vaccination to prevent cancer might work better than treating it after the fact, ya think?

Quadruple amputee soldier learns to adapt to life with transplanted arms.

‘Molecular movies’ will enable extraordinary gains in bioimaging, health research

Video

This is stupid, which means it made me laugh a lot.

What has been happening. . .

What has been happening. . .

And why I’ve been away so much. This started about three weeks ago, when my Mom went in to see the oncologist for her biopsy results, and the staff didn’t think that her shortness of breath and loss of consciousness was simply stress. They sent her to the emergency room, and I got a call that if I didn’t come to pick up my Dad, he’d be picked up by a long-term care facility and they might not release him to my Mom because she was so sick. Talk about incentive. I hadn’t finished doing laundry, so I went down with whatever I had to wear, my medications, and an aerobed.

Turns out, she’d been having these problems for a while (and, of course, downplayed them so nobody would be concerned) and it was a pulmonary embolism. A small one, but I don’t think size really matters much. Thank goodness they insisted. But here’s where the fun begins. You see, Dad has been declining mentally for several years, and his condition is another thing Mom has been downplaying. The reasons are numerous and complex, and I’m not going to get into too much detail because that’s outside of this narrative. Since Mom’s got atrial fibrillation and has been on blood thinners, which she stopped so she could get her biopsy (Non-Hodgkin’s lymphoma, treatable with chemo) it became kind of complex to get her clotting factor right at the same time as they broke up the clot in her lung – and while she was there, they wanted to install a chemo port and give her her first treatment.

This meant over a week in the hospital for her.

This meant over a week of caring for my Dad by myself.

At this point, my new meds had not kicked in, I was still having panic attacks, and I was trying to process a whole bunch of information without the benefit of Adderall, either. And my Dad’s dementia is. . .bad. Looking at the description of a seven-stage progression, he’s between five and six, and awfully close to entirely stage six. After just a couple of days with him, taking him back and forth to the hospital and then being yelled at later for not having told him Mom was in the hospital, not being able to do anything except have the same conversations with him over and over without him getting upset that I wasn’t engaging him, being awakened at all hours of the night and early in the morning either because he was wandering the house (sometimes on his way outdoors) or waking me up to ask where Mom was, I was really on edge. I texted my sister and asked if she could relieve me for the weekend, and, bless her heart, she showed up on Thursday night. Not only did it help me out enormously, but I now had someone else to corroborate my story about his condition.

Things were relatively OK, but then Mom called me on a Saturday to tell me that Dad was in the hospital – this was his second time, but his first one (a couple of weeks prior to Mom’s for the embolism) she didn’t tell me anything until he was home. This time, she really shouldn’t have been driving, but she would have if we hadn’t come down, so hubby and I headed down on Sunday to take her to visit Dad. We got her a wheelchair to take her around the hospital, because she needed it. Dad was really out of it, sleeping slouched in a chair when we arrived, so we went and got lunch. When we came back, nothing had changed, and Mom found that she couldn’t wake him up, that he muttered a few incoherent things, and we realized that his arms and legs were ice cold.

When we called the nurse and they realized that they couldn’t get his blood pressure, we were shooed out of the room, and soon there were more doctors and nurses than could even fit. They moved him to the bed, and tried to get a chest x-ray, but he was uncooperative and physically fighting them off. They figured that in addition to the one infection he had, he probably also had pneumonia, so they began an IV drip of antibiotics for that. He was conscious when we finally got back into the room, but nothing he said made any sense.

Once we knew he was out of danger (because we were pretty worried for a while) we brought Mom home. I called her the next day, and she had spoken to him, and he was doing better, but still thought he was being held in a jail for something. She had no idea if she actually had someone to give her a ride to her second chemo appointment, so I figured I would drive her myself and then go to the hospital to see if they could just care for him for a couple more days – because even this had not been enough for her to actually get a home health aide or a visiting nurse. I knew that there was no way that she could care for him and keep him out of danger while recovering from an infusion.

So this is where it gets dramatic.

I slept badly, of course, and set off a little before 6:30AM to pick her up. I went in with her to speak with the oncologist and we discussed, frankly, the reality that she would not be able to care for Dad by herself, and I think that hearing it from the doctor lent it credibility that it didn’t have coming from me. She agreed that as much as she wanted Dad to be able to stay at home with her, it was in her best interest that he be cared for around the clock somewhere else for at least a little while when she was feeling like crap.

I got Mom settled in with her pillow and blanket and book, and headed out to the hospital. I went to the nurses’ station, trying to keep out of his sight so I could speak to them without upsetting him. They shocked the heck out of me by announcing that they had been calling all morning because it was time to release him! I explained that this was really, really, really bad timing, because my Mom would be hooked up to chemo drips until at least 4PM, and I couldn’t take him to the oncologist’s office OR leave him home alone. Well, they told me, the papers had already been signed, so I’d have to take him and do one or the other. No room for negotiating.

The social worker was at the desk, and I asked her if he could stay. No. Are there any short-term places he can go? No, he doesn’t qualify for short-term rehab, so he’d have to go into long-term care, and then he’d be there permanently. I didn’t want to place him somewhere permanently, and I especially didn’t want to be the one responsible for placing him permanently, against my Mom’s wishes. I asked if I could speak to the doctor who signed the release. (n.b., at this point, my Dad has not seen me, but he no longer has a guard in the room – he’s all by himself, seated on a pad that sounds an alarm every time he gets up, at which point, nurses rush in and make him sit down again.)

The doctor comes out, and I have to say, I have not been treated so condescendingly or disrespectfully by a doctor in close to 20 years. I’m not naming the hospital or the doctor – I’m going to write to them, I don’t need them to have a bunch of people descending angrily upon them, because my anger should be just about all they can handle! I tried to explain to him that Mom was getting chemo all day, I live an hour and a half drive away, Dad’s dementia makes it impossible for her to care for him while she’s dealing with her own treatments, and isn’t there some way he can just keep Dad there for even one more day? He gives me the same line about either I take him home, or he gets committed permanently and there’s no way he’ll ever come home, and he’s a professional gerontologist and I should know that Dad’s mental condition will decline rapidly if he goes into a home and I’ll be responsible for giving him a death sentence.

That’s when I threw out the names of the other doctors with whom I had consulted who agreed that he needed nursing care while mom was sick (GP as well) and suddenly he’s all “Oh, I know them. Good doctors. Well, I wish you luck,” and then walked away with a smile as if he had not just implied that I don’t care if my father dies in a nursing home.

Believing I had no choice and needing to do something quickly, because Dad was beginning to get really angry with the alarms and the nurses and such, I arranged for a liaison from the home closest to my parents’ house to start the admission process. Dad had seen me at this point, so I had to sit with him for a bit, but I needed to call Mom and it’s impossible to make a phone call with Dad there because he gets upset if you’re talking but not to him. Of course, he tried to follow me out, and the alarm went off, and the nurses came, and he was fighting and yelling.

Down the hall, I tried Mom’s cell phone, but she didn’t have it or it wasn’t on, so I called the main number for the oncologist. The receptionist passed the message, and shortly after, I was talking to Mom about what I had been forced to do and why – then she passed the phone to one of the nurses and the oncologist’s social worker. They were pretty furious, because I’d been lied to. Yes, my only option was a nursing home, but it wasn’t a prison. We could take him out any time.

Trying to explain this to my Dad was an awful experience. He didn’t get the concept – of anything. He forgot who I was. He was angry because he’d been kept alone in this room for so long and didn’t understand why this alarm kept going off and why nobody would let him walk around. He was tired of waiting around to go visit whomever he thought he was visiting, because he didn’t remember that he was the patient. When the rep from the nursing home arrived, she was wonderful. It was obvious that she understood how to handle people with memory issues, and had the patience of a saint. She figured out the one thing that caught my Dad’s full attention – he wanted to take care of Mom. She told him that he was going to need to build up his strength so he could do that, so he was going to stay in this place and do physical therapy every day until he was ready to be Mom’s caregiver. And when she saw that I would tell him what was happening and then he’d get mad because nobody had told him this was happening, over and over again, she told me that the hospital social worker needed to see me so I should say goodbye to him. It got me out of the room, and Dad accepted it – but nobody actually wanted to talk to me. She just knew that was the only way to disengage.

I went back and stayed with Mom until her chemo was done. I hadn’t had anything to eat, so I was given some crackers and coffee by the oncology nurse. When the chemo was done, I went out to pick up Mom’s prescriptions while she packed clothes for Dad. I got back, and she was on the phone with Dad, explaining to him that he needed to get strong so he could take care of her. The moment they hung up, my sister called. I may have been a bit abrupt (sorry, Jen!) but I had been up since 4:30, had eaten nothing but those crackers since 6AM, and still had to drive to the home and drop off the clothes and pick up dinner from the diner.

The home was nicer than some, not as nice as others, but the staff was good, and nobody was restrained. Dad wanted me to take him on a tour, and this was just not an option at this point. Fortunately, a staffer was approaching us, and I asked her if she could show him around. She agreed, and I said my goodbyes. Picked up food. Ate. Drove home, got there about 10:30.

A couple of days later, I called Mom. She’s talked with Dad, and he thinks this place is pretty luxe, and he’s doing physical therapy so he can come home. She, meanwhile, has been able to sleep whenever she needs to and for as long as she wants, and get sick without Dad trying to “help” her. She didn’t want him to be taken away from home, but for the moment, things seem to be working out, and that takes a huge weight off my mind.

Learning from Research, The Results.

Learning from Research, The Results.

This is the part where my brain is going to explode. I might need to break this up into more than one post.

RESULTS
Preparation of HMECs

A single HMEC in its log phase was plated, and expanded to 1.4 × 106 to 1.5 × 106 cells (Fig.1). Plating efficiency during the two transfers of plates was 67 ± 0.9(mean ± SE)%. Based on these values, the number of cells that should have been produced at the time of harvest was calculated as 3.2 × 106(1.4 × 106/0.67/0.67). This value predicted that each cell harvested underwent 21.6 generations from the initial single cell. Doubling time was 48 h.

Strategy of cell culture. A single HMEC was inoculated in a well by limiting dilution, and the cell was expanded up to approximately 106 cells. Based on the plating efficiencies during the two transfers and the actual final cell count, the number of cells that should have been produced at the time of harvest and the number of generations observed were calculated. DNA was extracted from the final cells, and used for bisulfite sequencing. Six independent cultures were performed.
Slide1

HMEC – Human Mammary Epithelial Cells. They were put into a container, allowed to reproduce, and then they were checked to see if the right number of cells were made after specific numbers of generations. There were six containers of these cells. Once enough generations had passed and there were enough cells, their DNA was tested with the bisulfate test (illustrated in my earlier post.)

Gene Selection and Their Expression Levels

Methylation statuses were determined by bisulfite sequencing for CGIs in the promoter regions of the E-cadherin,p41-Arc, SIM2, 3-OST-2, and Cyclophilin A genes; CGIs in the downstream exon/introns of theE-cadherin, p41-Arc, and SIM2 genes; CpG sites outside CGIs of the E-cadherin and p41-Arcgenes; a NM-CGI of the MAGE-A3 gene; and differentially methylated region (DMR) of the H19 gene (Fig.2A). The former five genes were selected because they had CGIs in the downstream exon/introns that met a strict criterion of CGIs, regions of DNA of >500 bp with a G+C ⋝ 55%, and observed CpG/expected CpG of 0.65 (Takai and Jones 2002). The MAGE-A3 gene and the DMR of the H19 gene were selected as a representative NM-CGI and a region critically involved in genomic imprinting, respectively. By quantitative RT-PCR analysis, their expression levels were shown to range from almost none (SIM2 and MAGE-A3) to very high (E-cadherin), with p41-Arc, 3-OST-2 andCyclophilin A being intermediate (Fig. 2B).

Structures and expressions of the genes analyzed. (A) Schematic representation of the genomic regions analyzed. Regions analyzed by bisulfite sequencing are shown by closed boxes, and designations A–L correspond to panels in Fig. 3. CGI-P: a CGI in the promoter regions; CGI-outside: a CGI outside the promoter regions; Non-CGI: CpG sites outside CGIs; and DMR: differentially methylated region. All panels are drawn to the same scale. (B) Expression levels of the seven genes in HMECs.
Genome Res. 2003 May 13(5) 868-74, Figure 2

Sorry, I can’t even. All I know from this is that they looked at the results of the bisulfite sequencing and found what they were looking for – the methylation status in the CpG Islands from promoter regions of DNA stayed almost exactly the same. Unmethylated CGIs from non-promoter regions were more likely to become methylated. I’m afraid I don’t have the ability to explain this to you or tell how accurate or flawed it may be. I’m taking the researchers’ word on it. Correct me if I’m wrong.

Establishment of How to Measure MPERs

The CGI in the promoter region of the E-cadherin gene (Fig.3A), the non-CGI region of thep41-Arc gene (Fig. 3F), the CGI in the promoter region of theMAGE-A3 gene (Fig. 3K), and the DMR of the H19 gene (Fig. 3L) were found to contain two major populations of clones. The two major populations were considered to represent the methylation pattern of the two alleles in the original single cell. The methylation patterns of the two major populations were different from each other in the six cultures, which indicated that the HMECs before cloning had diverse patterns of methylation, but the patterns were relatively conserved during the culture from a single cell to approximately 106 cells. Therefore, we measured the number of errors in the methylation pattern based upon the culture from a single cell to approximately 106 cells. An MPER of a region in a culture was calculated from the number of errors in methylation pattern as described in Methods, and an average MPER of the region was calculated from the six MPERs obtained for the six cultures.

MPERS – Mammalian Protein Extraction Reagent
AlleleAn allele is one of two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. If the two alleles are the same, the individual is homozygous for that gene. If the alleles are different, the individual is heterozygous. Though the term “allele” was originally used to describe variation among genes, it now also refers to variation among non-coding DNA sequences.

So after making all those cells, they looked to see where and whether methylation status had changed.

Distribution of unmethylated and methylated CpG sites shown by bisulfite sequencing. Unmethylated and methylated CpG sites are shown by open and closed circles, respectively. (A)–(C) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the E-cadherin gene. (D)-(F) A CGI in the promoter region, a CGI outside the promoter region and CpG sites in non-CGIs of the p41-Arcgene. (G), (H) A CGI in the promoter region and a CGI outside the promoter region of the SIM2 gene. (I) A CGI in the promoter region of the 3-OST-2 gene. (J) A CGI in the promoter region of the Cyclophilin A gene. (K) A CGI in the promoter region of the MAGE-A3 gene, which is normally methylated. (L) A CGI in the differentially methylated region of the H19 gene.

Here’s where they found the differences:

Genome Res. 2003 May 13(5) 868-74, Figure 3

To examine the effect of an arbitrary selection of the “original methylation pattern” in ambiguous cases, a permutation test was performed for the CGI in the E-cadherin promoter region of HMEC10. One of the clones #5–#14 (Fig. 3A) was hypothesized as one of the original methylation pattern, and the number of errors in the methylation pattern was calculated. The numbers ranged from 18–22, and these values were expected to result in the average MPER ranging from 0.022–0.023. Similar permutation tests were performed for the CGI in exon 2 of the E-cadherin gene of HMEC12 and HMEC15. The numbers of errors in methylation pattern ranged from 13–16 for HMEC12 and from 12–15 for HMEC15, and these values were expected to result in the average MPER ranging from 0.050–0.058. These showed that arbitrary selection of the original methylation pattern in ambiguous cases does not seriously affect the resultant average MPER.

Some changes weren’t so cut and dried, so they checked those cases and found that they weren’t significant enough to change the findings.

The efficiency of bisulfite conversion was examined by analyzing DNA with no methylation in the CGIs in the promoter region and exon 2 of the E-cadherin gene. In the CGI in the promoter region, none of the 600 cytosines at CpG sites (30 CpG sites per clone, 20 clones analyzed) remained unconverted, showing that unconversion rate was almost 0 in this region under our experimental condition. In the CGI in exon 2, one of 483 cytosines at CpG sites (23 CpG sites per clone, 21 clones analyzed) remained unconverted, showing that the unconversion rate was 0.0021. These values showed that the MPERs in CGIs in the promoter regions are 10-fold more than the unconversion rates.

The bisulfate conversion was also tested separately for control to make sure the results would be valid in the experiment. This reinforced the finding that the promoter regions stayed stable.

MPERs and Fidelities of Methylation Pattern in the Genome

The average MPERs obtained for each region are summarized in Table1. Unmethylated CGIs in the promoter regions showed MPERs between 0.018 and 0.032 errors/site/21.6 generations. In contrast, CGIs outside promoter regions showed significantly higher MPERs, ranging from 0.037 to 0.091 (P < 0.01 or 0.005). MPERs in the CGIs outside the promoter regions were more than twice as high as those in the promoter regions of the same genes. MPERs in Various Genomic Regions

NM-CGI of the MAGE-A3 gene and methylated alleles of the DMR of the H19 gene showed MPERs of 0.002 and 0.007, respectively. Any genomic regions that were normally methylated, whether or not they were in CGIs, showed significantly lower MPERs than those unmethylated. This was particularly clear when the MPER of the allele methylated at DMR of the H19 gene was compared with that of the other unmethylated allele.

Interpretation of the tables, summary of findings.

This is not as good as part one, sorry. In other news, I couldn’t watch Besharam because it sucked, so I didn’t learn any Hindi, either. One more post to go in this series. Anyone who can clarify/explain better than I can, please comment – I’d appreciate it.

Learning from Research, Slowly and Methodically.

Learning from Research, Slowly and Methodically.

I was given a challenge on Twitter, and some people dismissed me as a failure because I didn’t have the academic background to come back with a quick answer. (I also discovered that I knew the answer, but forgot the words because of post-surgical anomia. I digress.) I find that this is a problem with a lot of people with certain types of expertise. They forget what it was like back when they were first learning, and no longer have the patience to explain. I don’t think it helps that there is a shit-ton of people on the internet spouting nonsense and being taken seriously. Naturally, some of them will assume that I’m doing the same, but I really don’t want to be lumped in with them, so I’m going to show them the process I go through, and how seriously I take learning new things and separating fact from fiction.

As I said in my previous post, you guys are wicked smart, and I am very often in awe of how much you know. But one thing you’re not so good at is communicating to people outside your fields of expertise. This is why we have bad science journalism. Ask Ed Yong. However, if you want to stop all your discoveries from degenerating into misrepresentation or woo, then you need people who can translate Science into English.

I was given a long, information-dense study, Fidelity of the Methylation Pattern and Its Variation in the Genome by Malcolm M. Campbell, so it’s going to take several posts to dissect, research, learn the background information, and try to explain it in an accessible way. I fully expect to be wrong several times, and encourage people to correct me – in such a way that ordinary people can “get it.” So here goes:

Abstract

The methylated or unmethylated status of a CpG site is copied faithfully from parental DNA to daughter DNA, and functions as a cellular memory. However, no information is available for the fidelity of methylation pattern in unmethylated CpG islands (CGIs) or its variation in the genome. Here, we determined the methylation status of each CpG site on each DNA molecule obtained from clonal populations of normal human mammary epithelial cells.

Methylation turns genes or pieces of genes “on” or “off”. There’s a detailed explanation of various ways it does this in the components of the whole process from DNA to cell, but it’s kind of hard to understand if you haven’t done a lot of reading beforehand. I’ll give you the link anyway.

CpG sites – the quick and dirty Wikipedia definition is this: The CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. If you don’t remember from your Biology classes, or your biology classes never taught you, your entire DNA strand consists of combinations of four nucleotides – Cytosine, Guanine, Taurine, and Adenosine. I’m not going to get into that right now, because it’s just going to confound this with too much information, but if you think about the movie “Gattaca,” you’ll notice those four letters. In a movie about genetic engineering. Because those are the four letters you see in an illustration of a piece of DNA. The researchers were looking at the parts where the cytosine and guanine were next to each other.

Specifically, they were looking at epithelial cells from normal breast tissue. The link may be a little difficult to understand, but I think if you read all the way through, you’ll at least understand some of the reasons these cells were chosen. They have a lot of unique characteristics, and they’re pretty tough.

So the idea here is that we already know that if the cytosine and guanine pair are methylated in the on position or the off position in the DNA, that they’re going to stay that way in the cells that are produced by those instructions from the DNA. What we don’t know is that if that pair is unmethylated, will the cells made from the DNA instructions also be unmethylated? IOW, if they’re not already told to be switched on or told to be switched off, will they still be in that “neutral” position? In order to test that, they took a bunch of those epithelial cells and tested each one to see if it was methylated or unmethylated so they could get them to reproduce and see what happened.

This illustration is not specific to this piece of research, but keep reading, and you’ll see how it relates.. I wanted to give you a visual aid in case you learn better that way.

Methylation pattern error rates (MPERs) were calculated based upon the deviation from the methylation patterns that should be obtained if the cells had 100% fidelity in replicating the methylation pattern. Unmethylated CGIs in the promoter regions of five genes showed MPERs of 0.018–0.032 errors/site/21.6 generations, and the fidelity of methylation pattern was calculated as 99.85%–99.92%/site/generation. In contrast, unmethylated CGIs outside the promoter regions showed MPERs more than twice as high (P < 0.01). Methylated regions, including a CGI in theMAGE-A3 promoter and DMR of the H19 gene, showed much lower MPERs than unmethylated CGIs. These showed that errors in methylation pattern were mainly due to de novo methylations in unmethylated regions. The differential MPERs even among unmethylated CGIs indicated that a promoter-specific protection mechanism(s) from de novo methylation was present.

This explains how they figured a reasonable range of variation. The “islands” of unmethylated cytosine/guanine pairs in five genes over 21.6 generations (this is statistics, not absolute numbers. You clone enough cells, you sure as heck can get six tenths of a generation.) stayed unmethylated most of the time. This came from promoter regions, which are the areas in DNA that call the shots. It’s more likely that instructions from promoter regions are going to be followed.

The unmethylated cells that didn’t come from promoter regions showed more deviations – the cells after several generations were twice as likely to be different from the originals than the ones that came from the promoter regions. The methylated cells, which, as I mentioned, already have the specific instructions to turn a gene on or off, were more likely to maintain their integrity even if they weren’t from promoter regions. The unmethylated cells didn’t’ have that instruction, and hadn’t been told to stay unmethylated (because they weren’t from promoter regions) and so they just did whatever seemed right at the time and, well, mistakes were made.

CpG methylation is known to serve as cellular memory, and is involved in various biological processes, such as tissue-specific gene expression, genomic imprinting, and X chromosome inactivation (Jones and Takai 2001; Bird 2002; Futscher et al. 2002;Strichman-Almashanu et al. 2002). These important functions of methylations are based upon the fact that the methylated or unmethylated status of a CpG site is faithfully inherited. The methylated status of a CpG site is inherited upon DNA replication by the function of maintenance methylase, represented by DNA methyltransferase 1, which is located at replication forks and methylates hemimethylated CpG sites into fully methylated CpG sites (Leonhardt et al. 1992; Araujo et al. 1998; Hsu et al. 1999). The unmethylated status of a CpG site is inherited by not being methylated upon DNA replication or any other occasions. Unmethylated CpG sites generally cluster to form a CpG island (CGI), and most CGIs are kept unmethylated (Gardiner-Garden and Frommer 1987; Bird 2002). Methylations of CGIs in promoter regions are known to cause transcriptional silencing of their downstream genes by changing chromatin structures and blocking transcription initiation (Bird 2002;Richards and Elgin 2002). There are limited numbers of CGIs that are normally methylated (normally methylated CpG islands; NM-CGIs) (De Smet et al. 1999; Futscher et al. 2002). CpG sites outside CGIs, especially those in repetitive sequences, are also normally methylated (Bird 2002).

CpG methylation is important. It is carried on pretty faithfully when cells reproduce. It’s also important that unmethylated CpG remains unmethylated, and that’s usually passed on to new cells as well. Most of the unmethylated sites form a cluster called a CpG Island, or CGI. If these unmethylated CGIs become methylated, then it changes what genetic instructions get turned on or off in future generations of cells, if they’re in promoter regions. But it’s not always bad for CGIs to be methylated, because sometimes that’s on purpose.

I’m going to hold off on the transcription and chromatin stuff for later, because I think it’ll stick better when the paper goes into more detail.

To keep the methylation pattern, maintenance of both methylated and unmethylated statuses of CpG sites during DNA replication is necessary. However, the fidelity of the methylation pattern has been analyzed only for the maintenance of the methylated status (Wigler et al. 1981; Otto and Walbot 1990; Pfeifer et al. 1990). The fidelity in maintaining the methylated status of an exogenously introduced DNA was shown to be 94% per generation per site by Southern blot analysis (Wigler et al. 1981). The fidelity in maintaining the methylated status of a CGI in the 5′ region of the PGK1 gene, which was derived from the inactive X chromosome, was estimated to be 98.8%–99.9% per site per generation by the ligation-mediated PCR method after chemical cleavage of DNA (Pfeifer et al. 1990).


We’ve already studied methylated CpG sites and found that it’s pretty consistent. Some studies attesting to that are cited. We know that keeping them unmethylated is also important, but that hasn’t been investigated to our satisfaction.

Normally unmethylated regions might show different fidelities from normally methylated regions. Even among the unmethylated CGIs, the fidelities of their methylation pattern have been suggested to be different according to their location against a gene promoter. Methylation of CGIs in promoter regions almost always leads to transcriptional silencing while that of CGIs outside promoter regions does not (Gonzalgo et al. 1998; Jones 1999). Considering the cellular expense in maintaining methylation pattern, a cell could sacrifice the fidelity of methylation pattern for CGIs outside promoter regions. In addition, by recent genomic scanning techniques for methylation changes (Ushijima et al. 1997; Toyota et al. 1999; Costello et al. 2000; Jones and Baylin 2002), aberrant methylations of CGIs in cancers are observed in a nonrandom manner (Toyota et al. 1999; Costello et al. 2000; Kaneda et al. 2002a; Kaneda et al. 2002b). It is indicated that CGIs outside promoter regions were more frequently methylated than those in promoter regions (Nguyen et al. 2001; Takai et al. 2001; Kaneda et al. 2002a; Asada et al. 2003).

Unmethylated CGIs are more likely to change than methylated ones. Unmethylated CGIs from promoter regions of the DNA pretty consistently shut down the things they’re supposed to shut down, exactly as planned. Unmethylated CGIs from outside promoter regions of the DNA are not so good at that – they’re more likely to become methylated when they’re supposed to stay unmethylated. Some of this methylation of unmethylated CGIs has been seen in cancer. So that’s one example of why we don’t want this to happen.

Here, we analyzed the methylation status of each CpG site on each DNA molecule by the bisulfite sequencing technique (Clark et al. 1994) in six clonal populations of normal human mammary epithelial cells (HMECs), for CGIs in the promoter regions, CGIs outside the promoter regions, and CpG sites outside CGIs. By analyzing the deviation from the most common two patterns, MPERs, which reflected the fidelity in replicating both methylated and unmethylated statuses, were measured.

Like a five-paragraph essay here. Restating what they’re going to do and how they’re going to do it. Remember the illustration? Bisulfite sequencing technique. (Really detailed explanation, Wikipedia explanation).

And now my brain is very, very tired. I am going to watch “Besharam” because I’m also trying to learn Hindi, and I might as well be looking at Ranbir Kapoor while I’m doing it. Heh. I will continue this in a later post. Feedback is welcome and encouraged.

Science People!

Science People!

I’ve been getting a lot of attention on Twitter for the last couple of posts, and that’s given me a lot of articles to read, blogs to keep up with, and Twitter users to follow. Some people got a little testy, and I don’t blame them, because they know more than I do. I get it.

Let me tell you something right now. I am not a professional scientist. I got my Bachelor’s degree in Spanish Language and Literature back in the early 80s, and distanced myself from science since I had to take my only B-track class in all of High School in Biology. I didn’t get it, I didn’t see the point, I put no effort in, and I sucked at it.

That’s ADHD.

But then I started reading books about the brain, and that struck a chord with me because my brain is not the nice neurotypical model. I started reading blogs and websites about the brain, and medicine, and genetics. I learned how to read published research (and occasionally got friends who would sneak me links to full text articles) and would search in the middle of searches when I found terms I didn’t understand or biological processes or mechanisms that were new to me but essential to understanding what I was reading.

This obsessive pursuit of information is also ADHD, BTW.

This means that there are gaps in my knowledge. I am not ashamed to admit that you know more than I do. Please don’t get angry with me when I’m wrong – explain to me why I’m wrong and then tell me how to understand it the right way. I don’t want to be right to win arguments or lord it over people, I want to be right because I have the correct information. You can help me with that.

Thing is, one thing I know I’m really good at is teaching other people things. I take my mistakes, the process by which I figured something out, and the way it works at the most basic level, and try to use that to explain what I know in a way so that other people can “get it.” There are several college students out there pursuing degrees in science because I got them all excited about it. They’re getting the chance I missed out on.

So, you want more minions? (MUHAHAHAHA!!) Give me comments. Help me understand. Because if you help me understand, I can help other people understand. I’m an intelligent woman, I’ll get it pretty quickly, and when I don’t, I’m not in the least ashamed to admit that I was wrong. We can have a mutually supportive and respectful interchange, and I’ll do my part to explain things in an accessible way, using the tools you give me.

Really. Comment. email. Bring it on. I love you guys!

Your Inner Fish

Your Inner Fish

I loved this book, and now PBS is making a miniseries with Neil Shubin. I can’t wait.

A long time ago, right after I read it, I put up a series of posts on a forum detailing the wonderful things I had learned from it. After a while, the threads were hijacked by people who just didn’t get it – or didn’t want to get it – and they disappeared into obscurity. But I stand by what I wrote, and now that this book is back in public view, I want to share these thoughts again. This is a long read, over 4,000 words, and it’s taken from a forum thread, so there are parts that don’t flow entirely well, but I don’t want to edit or rewrite it because it captures the wonder and excitement I felt when I first read the book and I don’t want to change that.

So settle down with a nice cup of tea if you’re ready to go below the fold.

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10 Things I Have Learned About Abortion from Pro-lifers.

10 Things I Have Learned About Abortion from Pro-lifers.

1. Women choose to have sex. Men are apparently not involved in this decision-making process.

2. Women who do not use birth control are irresponsible and should never have sex.

3. Women who use birth control are also irresponsible, because they know that birth control is not 100% foolproof and should never have sex.

4. Being pro-life has absolutely nothing to do with religion. It’s just a coincidence that my God is opposed to abortion, and if yours isn’t, then you’re worshiping the wrong God.

5. No matter how many examples you find of God-sanctioned infanticide in the Bible, it in no way indicates that God is OK with baby-killing. Baby-killing on his orders is OK because reasons. If he says it’s OK, it’s OK, but he definitely didn’t say abortion was OK except in the parts where he did.

6. All the aborted babies could have gone on to do great things. None of the aborted babies would have been “welfare queens” or criminals or deranged genocidal dictators.

7. People are lined up to adopt babies. If you give your baby up for adoption, it will find a loving family. It definitely, positively, won’t join the half million kids already available for adoption or be one of the 23,000 who age out of the system without being adopted every year. Oh, and it will be happy with its family, who will never turn out to be abusive in any way.

8. It is never OK to abort a baby that resulted from consensual sex. Conception circumstances are paramount, which is why it’s OK to abort rape babies. Consensual sex babies are alive at the moment of conception because of consent. Rape babies are alive at the moment of conception, too, but it’s OK to abort them because they aren’t the consequences of the choice of an irresponsible woman. Don’t ask me to explain this, I’ve tried and tried and still don’t get it.

9. If abortions are illegal, nobody will need them. Only 1% of all abortions are for high-risk situations like the life of the mother or significant defects in the fetus, and letting women die and having babies who are severely handicapped (even if they’re guaranteed to die after birth) is a risk that people who aren’t dealing with these situations are willing to accept.

10. Even if you are too poor to support a child, too young to be a parent, too ill mentally or physically to be a parent, addicted to drugs and unemployed and homeless, married to an abusive spouse or a pedophile, the baby is a gift from God and all your problems will go away as long as you don’t get an abortion.

ADHD and Pharmaceutical Fearmongering.

ADHD and Pharmaceutical Fearmongering.

It’s never difficult to find articles about how ADHD is some trumped-up condition made up to excuse poor behavior and/or line the pockets of the medical industry. Whether the writer assumes one or both of these, it’s necessarily bound together with denial, ignorance, and hyperbolic claims. Sometimes all you can do is get angry, but other times the writer gives you a chance to deconstruct his points. The Price of ADHD Business is that second kind.

He opens with this blockbuster:

Over 12 million children and young adults consume ADHD stimulant and psychiatric medications in the United States. Pharmaceutical corporations generated near 9 billion dollars in 2012 for ADHD stimulant drug sales, representing 5x the 1.7 billion in sales ten years ago.

Shocking, isn’t it? Except that in the US, the Pharmaceutical Industry makes about $345 billion a year. That means that psychiatric medications make up a whopping 2.6% of the bottom line. Hardly one of their biggest players, compared to drugs for cholesterol, pain management, and cancer treatment, which are much better performers when it comes to percentage of business. Also, notice the subtle slide from “ADHD stimulant and psychiatric medications” to “ADHD stimulant drug sales,” because this will be important.

More alarming, this rate of consumption represents 3x the world’s children combined, according to data collected by Scientific American. The business model of behaviorally assessing and prematurely medicating young school age children with powerful stimulant and psychoactive drug therapy for over 40 years is now under fire. The Government Accountability Office (GAO) Child Foster Care Drug Audit Report uncovered dangerous and unethical prescribing practices. Widespread abuses of overmedicating young foster care children with ADHD stimulant as well as psychiatric medications prior to ruling out nutritional, physiological, and environmental risk factors were uncovered by the largest child foster care prescription drug audit in American history.

Of course, being a first-world country, it’s more likely that we have 3x the children being treated for, say, cancer or juvenile diabetes, or any number of other childhood diseases, so there’s something of a leap from claiming that 3x the children being treated means that something is being treated too much – or “prematurely.” I notice also that the source of the alarmist rhetoric comes from a study of children in foster care. Well, this is a problem with foster care, not with all children. Foster care children are more likely to have disabilities, both physical and mental, meaning they’re more likely to actually need treatment. They’re also covered by state medical programs that make it pretty easy to get treatment that a self-payer parent might not be able to obtain. There’s more, but if a mere scratch on the surface can reveal that we’re comparing apples to oranges, there’s not much need to go even deeper.

In today’s America parents, educators, and prominent healthcare professionals challenge the 40-year ADHD business model, as the ADHD diagnosis rate surpasses epidemic status in 2014. The symptoms of ADHD are real and in many cases can be debilitating to children as well as adults. Especially in the young child population, the ADHD business model of assessment and treatment requires immediate reform. Children have a right to receive comprehensive bio-assessments as well as behavioral assessments to determine cause of their symptoms prior to powerful stimulant and psychoactive drug therapy.

Yes, we have a serious epidemic of about 5-8% of the population. That’s massive. Not. Notice how he snuck in the disclaimer (like, some of my best friends have ADHD!!!) but still calls the diagnosis and treatment of ADHD a “Business Model.” I sense a broken irony meter. Also, have you ever taken any of these medications? The stimulants are among the least powerful ones out there, with the most immediate effect (no two-month waiting period) and little to no withdrawal problems. Lumping them in with all psychoactive medications is disingenuous, especially for someone banking on his rep as a Pharmacist.

The Diagnostic and Statistical Manual for Mental disorders (DSM) lists ADHD as a mental disorder. The DSM diagnosing criteria, created by psychiatrists, involves a subjective behavioral assessment process which forces children primarily into premature drug therapy. Although seven out of ten children may exhibit an initial positive behavioral response to stimulant drug therapy for focus and attention, the long term side effects are now known.

Yes, they are, and they’re not terribly scary. Children who start using methylphenidate or dextroamphetamine medications may grow up to be as much as one centimeter shorter than their peers. Of course, they’ll also grow up happier and more successful and less likely to abuse drugs than their non-medicated ADHD peers, but that runs counter to the narrative here.

The Johns Hopkins Child Center Study results of 2013 prove that stimulant drug therapy should not be the primary intervention in young children. This study followed four year old preschool children who were diagnosed by their physicians for ADHD and medicated with stimulant drug therapy for a six year period. When the ADHD assessments were reviewed at age ten, over ninety percent of the children were worse off in their condition. Long term side effects of ADHD stimulants may include anxiety, minor depression, as well as aggressive behavior. Additionally, the Hopkins study determined that ADHD causes an economic burden to the US exceeding 45 billion dollars, annually.

To the first sentence I say, “Well, duh.” The primary intervention should be behavioral, with medications added to supplement as needed. This is not news, and it does not run counter to what any Medical Association is recommending, even the American Psychiatric Association, which says “Behavioral therapy and medication can improve the symptoms ofADHD. Studies have found that a combination of behavioral therapy and medication works best for most patients.” Add to this yet another lie, because what the Johns Hopkins study revealed was (prepare to be shocked) that ADHD doesn’t go away, and medications don’t cure it, just relieve the symptoms while they are in effect. And the economic burden he’s talking about? That relates to the consequences of untreated ADHD – people in jail, people who are substance abusers, people who are unable to work, people who have other health issues that are related to ADHD.

Many parents are not aware that a diagnosis of ADHD for their child is a diagnosis for mental disease in accordance to the DSM. Once a young child is placed on ADHD stimulants including Adderall or Ritalin prior to ruling out causative risk factors, there is an increased health risk. Additional medications for the treatment of long term side effects may be required due to the development of other behavioral symptoms.

Actually, parents know this, because lots of the evaluations are related to getting assessments for school. And it’s not the stimulants that increase the risk of further diagnoses and additional medications as much as the fact that ADHD is usually not alone, and the co-morbid conditions are discovered because the children are being observed and treated by doctors. The medications don’t produce these problems.

For example, the GAO drug audit uncovered a 2,200 percent increase in drug expenditures for atypical antipsychotic medication reimbursement to the state of Michigan during an eight year period from 2000 to 2008. Children in foster care, as the report states, were abusively prescribed powerful antipsychotic medications including Abilify, Zyprexa, Seroquel, Geodon and Risperdal. The Michigan Medicaid system was billed an increase of 40 million dollars during an eight year period just for this one class of medications in foster care children. US Senator Thomas Carper, requestor of the GAO drug audit and chairman of the Homeland Security & Government Affairs Committee, stated “I was almost despondent to believe that the kids under the age of one, babies under age one, were receiving this kind of medication”.

Remember what I said about the creep from the “all psychiatric medications” to “ADHD stimulant medications”? Here we go with another false equivalency. Antipsychotic medications are the last resort, used for treating not just ADHD, but ADHD with serious comorbids that would make the children a danger to themselves or others. Also, remember that this is the foster children, not all children with ADHD as a whole. AND keep in mind that several of these antipsychotics are essential for the treatment of schizophrenia and seizure disorders, which are probably too legitimate to mention in the context of this article. So the figures on antipsychotics for foster children in one state is cherry-picked data that in no way reflects that there is an epidemic of children with ADHD receiving inappropriate medications.

Should ADHD be labeled a mental disease especially in young children who have not been given the right to find the cause of their symptoms prior to stimulant drug therapy? Or, should ADHD be classified as a symptom of condition with underlying causative nutritional, physiological, and environmental risk factors?

Um, yeah, it should. Get inside our heads, mister – it’s definitely a mental disease. It’s certainly not something we can choose or turn off at will. And the cause of their symptoms is mental – the other “causative” factors have been thoroughly debunked as “causes” in study after study. So this is a giant flaming strawman.

The German magazine, Der Spiegel, quoted a prominent American ADHD psychiatrist in their February 2, 2012 issue. Dr. Leon Eisenberg, who coined the term ADHD over forty years ago, stated “ADHD is a prime example of a fictitious disease.” At age 87, this was Dr. Eisenberg’s last interview prior to his death. During the last forty years, he was involved in pharmaceutical trials, research, teaching, as well as the development of social policy pertaining to child psychiatry. He was a recipient of the Ruane Prize for Child and Adolescent Psychiatry Research. Currently, over fifty percent of psychiatrists on the DSM panel responsible for ADHD diagnosing and treatment protocols have direct business ties to drug manufacturing corporations.

All that education, and Mr. Granett doesn’t know how to check snopes. How sad. As to that second claim, well, a link would be nice, but I’m not surprised it’s absent, since the actual ties would be openly disclosed and not as incriminating as the author would like them to be.

Dr. Thomas Insel, Director of National Institute of Mental Health, stated on April 29, 2013 “patients with behavioral conditions deserve better… the current assessment process lacks validity.” He supports research that better treats and may even prevent the development of behavioral symptoms in children.”

Again with a diversionary link. Psych Central has some validity, but “The Verge”? Really? Why not link to the NIH’s Research Domain Criteria which explains that what this means is that the NIH wants to have research focused on multidimensional approaches to research, and research that is targeted towards evaluation of symptoms and behaviors rather than whole conditions, because we now know that there’s a lot of crossover and and a narrower approach will produce more successful and useful research. Oh, but that would not support the POV of the author. That’s OK, now you can see what Insel was really talking about. You’re welcome.

ADHD symptoms can be reversed through a process of differential diagnosing. The elimination of nutritional, physiological and environmental risk factors prior to premature drug therapy is the new ADHD Business Model for helping children and adults reclaim their behavioral and mental health. The Action Plan for Childhood Behavioral Conditions discussed in the book Over Medicating Our Youth as well as the upcoming 2nd edition T he American Epidemic: Solutions for Over Medicating Our Youth provides critical bio-assessment information to find the cause of ADHD symptoms. This action plan provides an informational template to unite parents, teachers as well as all healthcare professionals for the purpose of helping children win the battle against behavioral challenges.

Many assessments help determine the cause of ADHD symptoms. Learn how bio-assessments for reactive hypoglycemia, diabetes, the brain-gut connection, cervical spinal alignment, exercise, whole food nutrition, brainwave optimization, and nutritional enzyme supplementation may reverse ADHD symptoms.

And now, ladies and gentlemen, we begin our final descent into woo. ADHD symptoms cannot be reversed by any of these things, and. . .OMG, this whole thing is an advertisement for. . .wait for it. . .a book co-authored by Frank J. Granett! It’s so good that he had to cite himself! And real medical terms weren’t sufficient, so we have to make up some that sound really sciency, like “differential diagnosing,” and “reclaim their behavioral and mental health,” and “bio-assesment” so we can sell ineffective treatments to gullible patients. Blood sugar problems can be diagnosed and treated without ADHD medications. The “brain-gut connection” has no supportive research except in patients with full-blown Celiac Disease. Cervical Spinal Alignment is Chiropractic’s uglier younger brother, even less useful than regular Chiropractic for treating anything, much less neurological conditions. “Brainwave Optimization,” don’t even get me started. As for the rest, we already touched on how none of these things are causative, so they are not going to be curative.

But this is what it usually comes down to, isn’t it? The voices that protest the loudest that ADHD is a fake disease created to make money by the pharmaceutical industries tend to end up thinking that it’s real enough to be treated by whatever they themselves are selling. If only there were an all-natural cure for hypocrisy. . .

Treating the Symptoms, and the Placebo Effect

Treating the Symptoms, and the Placebo Effect

Proponents of various forms of alternative medicine regularly rally under the claim that medicine treats only the symptoms, while their favored modality “treats the whole person.” I’ve long known that this is wrong, and I could enumerate all the reasons why, but only now did it occur to me that there’s an even deeper level to this inaccurate claim that I haven’t seen addressed elsewhere – irony.

I’m not going to try to get into so much detail that it obscures the point (for a change) so I’ll stick to the examples that directly apply to my inspiration. Doctors and scientists who blog cover the overt falsehoods that are relative to their specialties with far greater specificity than I ever could. They can even tell you how each individual CAM treatment doesn’t work and why. I don’t think I need to do that, because I could never attain that level without the education, experience, and dedication that these science-based medicine bloggers have.

Instead, I’m going to draw from my own experience at a forum in which we discuss mental disorders – ADHD in particular, but also its other delightful companions and complications – where alternative treatments are accorded an undeserved level of respect and science-based medicine is treated with derision. In this place, since we are dealing with conditions that are complex in origin and difficult to reproduce and test in animal models, speculation is going to be a given. However, much of the speculation involves disregarding or even discarding the huge volume of information we already have from research.

There is absolutely no question that each condition being discussed is brain-based. There is absolutely no question that any effective treatment for these conditions is going to have to be a treatment of the brain. And there is absolutely no question that all the current approaches are aimed at relieving symptoms, whether through medication or other therapies, because research on the cause of symptoms yields results much more quickly than research looking at the most complex organ of the human body will yield information on causes. Science is churning away at brain research; new tools and knowledge are helping it to advance more quickly than it did in the past, and the findings from these are being used to develop even better tools and knowledge. Still, because there are practical and ethical limits on researching living human brains, results will not come as fast as they do for other diseases and conditions that involve other organs with simpler functions than the brain has.

Now that the introduction is out of the way. . .
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Chiropractic Cures Nothing

Chiropractic Cures Nothing

There’s an interesting idea out there among people who adhere to a belief that can be proven to be less than substantial that in order to contradict or challenge that belief, one must become an expert in that belief. It’s silly, and it’s frustrating to run into. It’s also usually hypocritical, because people who are firm believers in something do not apply the same standards to themselves – and in this particular case, the folks who are insisting that one must become an expert in the workings of chiropractic before being qualified to dismiss them feel no such obligation to become expert in the voluminous amount of medical knowledge that provides robust evidence for the failure of chiropractic. I mean, you’re presenting me with a book about how chiropractic can fix an area of the brain. . .if I have to learn all about chiropractic to say it doesn’t work, how come you don’t have to become an expert in neurology to tell me that the neurological impairment evidence is wrong? (The first place I saw this argument was coming from Christian Apologetics. . .who didn’t, BTW, become experts in any other religions before declaring that they were immune from criticism by anyone without a degree in Biblical Theology. . .)

The flaw in the argument is that you really don’t need to be an expert in something to know it’s bogus if there’s good, solid information that it couldn’t possibly work and/or it’s making ridiculous claims in the first place. I could be picking anything to poke at right now, but because the thing that’s irritating me right now is ridiculous claims about chiropractic and being told to STFU until I become an expert in chiropractic, that’s what I’m gonna talk about.
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